This study was aimed at characterizing the ultrastructural morphology of the normal acetylcholine (ACh) innervation in adult rat parietal cortex. After immunostaining with a monoclonal antibody against purified rat brain choline acetyltransferase (ChAT), more than 100 immunoreactive axonal varicosities (terminals) from each layer of the Par 1 area were photographed and examined in serial thin sections across their entire volume. These varicosities were relatively small, averaging 0.6 micron in diameter, 1.6 microns 2 in surface, and 0.12 micron 3 in volume. In every layer, a relatively low proportion exhibited a synaptic membrane differentiation (10% in layer I, 14% in II-III, 11% in IV, 21% in V, 14% in VI), for a I-VI average of 14%. These synaptic junctions were usually single, symmetrical (> 99%), and occupied a small portion of the surface of varicosities (< 3%). A majority were found on dendritic branches (76%), some on spines (24%), and none on cell bodies. On the whole, the ACh junctional varicosities were significantly larger than their nonjunctional counterparts, and both synaptic and nonsynaptic varicosities could be observed on the same fiber. A subsample of randomized single thin sections from these whole varicosities yielded similar values for size and synaptic frequency as the result of a stereological extrapolation. Also analyzed in single sections, the microenvironment of the ChAT-immunostained varicosities appeared markedly different from that of unlabeled varicosity profiles randomly selected from their vicinity, mainly due to a lower incidence of synaptically targeted dendritic spines. Thus, the normal ACh innervation of adult rat parietal cortex is predominantly nonjunctional (> 85% of its varicosities), and the composition of the microenvironment of its varicosities suggests some randomness in their distribution at the microscopic level. It is unlikely that these ultrastructural characteristics are exclusive to the parietal region. Among other functional implications, they suggest that this system depends predominantly on volume transmission to exert its modulatory effects on cortical activity.